The present invention relates to the discovery of novel genes encoding a polypeptide having creatinine deiminase activity and methods of use. Also disclosed is a kit using the novel sequences for determining the concentration of creatinine in a sample.
The synthesis of creatinine is a multi-stage process that occurs in several different organs of the mammalian body, cf. FIG. 1 (Narayaman, S., Appleton, H. D. (1980). Clin. Chem. 26: 1119–1125). In the kidneys, omithine and guanidine acetate are produced from the amino acids arginine and glycine, respectively. Once formed, omithine and guanidine acetate travel from the kidneys, over the bloodstream, to the liver. Here, guanidine acetate is converted to creatine, and subsequently transported and distributed over the body, including to the musculature. In the muscular tissue, creatine can then undergo phosphorylation by creatine kinase (EC 2.7.3.2.) to create the high-energy molecule, creatine phosphate. Creatine phosphate functions as an important power supply for muscle tissue, by providing a ready source of reserve phosphate for the continuous regeneration of ATP during the fast bypass process of muscular energy production. During the recovery phase of muscle energy regeneration, creatine can be rapidly rephosphorylated to produce creatine phosphate. Typically, creatine phosphate is rapidly and continuously degraded to form free creatinine and inorganic phosphate.
The elimination of creatinine from the body is primarily performed by the kidneys, once the creatinine encounters the renal glomerular filtration apparatus. The elimination of creatinine takes place in a constant relationship with respect to overall muscle mass and body mass. When this relationship is compromised an increase of creatinine concentration in the plasma can result, indicating a possible disruption in muscle and/or kidney function.
Endogenously formed creatinine is typically neither reabsorbed nor excreted in the kidneys when creatinine metabolism is functioning properly. As such, measuring creatinine concentration in blood plasma could be a basis for diagnosing renal dysfunction. However, plasma creatinine concentration considered alone does not provide significant diagnostic sensitivity, thus its measurement is insufficient to evaluate kidney function, and in particular, the glomerular filtration rate of creatinine.
A preferred indicator for the status of the creatinine glomerular filtration rate, sufficient for clinical interests, is the measurement of endogenous creatinine clearance, whereby a determination of the creatinine concentration can be made in a sample of body fluid, including plasma and urine. Several clinical scenarios where determining the rate of endogenous creatinine clearance can be diagnostically useful include measuring a compromised glomerular filtration rate, discovering the presence of pathological urine components, hypertonia, ascertaining the status of chronic kidney patients, progress of hemodialysis treatment, metabolic disturbances, pregnancy, or medications producing potentially nephrotoxic metabolites.
Determining the creatinine concentration in the plasma and urine relies on several important chemical principles and procedures including colorimetric procedures for confirming and evaluating creatinine presence.
One common procedure used for detecting creatinine concentration in a sample is a colormetric reaction provided by the Jaffé method, whereby creatinine reacts with picric acid in an alkaline environment, to form a yellow-reddish complex (i.e. a Jankovski complex) (Jaffé, M. (1886) Z. Physiol. Chem. 10: 391–400), which is measured photometrically using a wavelength of 500–550 nm. One notable disadvantage of this procedure is its known nonspecificity, as numerous non-creatinine chromogenes, including bilirubin, glucose, ketone bodies, acetoacetate and pharmacons such as cephalosporine and metamizol can also form a Jankovski complex upon reacting with alkaline picrate (Soldin, S. J., Henderson, L., Hill, J. G. (1978). Clin. Chem. 26: 286–290; Kroll, M. H., Hagengruber, C., Elin, R. J. (1985). J. Biol. Chem. 115: 333–341; Swain, R. R, Briggs, S. L. (1977). Clin. Chem. 23: 1340–1342; Saah, A. J., Koch, T. R. Drusano, G. L. (1982). JAMA 247: 205).
To overcome the nonspecificity problems associated with the Jaffé method, scientists have made numerous attempts to improve the specificity in detecting the creatinine-picrate chromophore. For example, one attempt consisted of absorbing creatinine to Fullererde in order to accurately determine the creatinine concentration (Knoll, E., Stamm, D. (1970). J. Clin. Chem. Clin. Biochem. 8: 582–587; Knoll, E., Wisser, H. (1973). Z. Klin. Chem. Kin. Biochem. 11:411). Other efforts to determine creatinine concentration have included the use of an autoanalyzer of the “continuous flow generation”, whereby a dialyzed sample is used for analysis. Such a sample was thought to reduce interference from external factors and competing substrates which could otherwise increase the error rate of the creatinine measurement (Popper, H., Mandel, E. Mayer, H. (1969). Biochem. Z. 291: 394; Scheuerbrandt, G., Helger, R. (1969). Aertztl. Lab. 15: 65).
A further known creatinine detection procedure includes reacting creatinine with o-nitrobenzaldehyde, whereby creatinine is degraded to methylguanidine and measured using the Sahaguchi reaction (Van Pilsum, J. F., Martin, R. P., Kito, E. Hess, J. (1956). J. Biol. Chem. 222: 225–236).
Another well known procedure for detecting creatinine involves reacting creatinine with 3,5-dinitrobenzoic acid and/or 3,5-dinitrobenzoylchloride to form a magenta-red complex, which is measured photometrically (Langley, W. D., Evens, M. (1936). J. Biol. Chem. 115: 333–341; Benedict, S. R., Behre, J. A. (1936). J. Biol. Chem. 114: 515–532; Sirota, J. H, Baldwin, D. S., Villareal, H. (1950). J. Clin. Invest. 29: 187–192).
None of these procedures shows improved specificity for ascertaining creatinine concentration when compared to the colorimetric measurement provided by the Jaffé method.
There are several different types of enzymatically-based procedures generally available to determine the creatinine concentration in a sample. In two of these procedures creatinine is converted in a first step to creatine using creatininase (EC 3.5.2.10).
One known procedure involves multiple enzymatic steps. For example, creatine is first converted to creatine phosphate via creatine kinase (EC 2.7.3.2.) to produce ADP, which in the presence of PEP and pyruvate kinase (EC 2.7.1.40) forms pyruvate and ATP, e.g., FIG. 2. The pyruvate is then converted to lactate in the presence of lactate dehydrogenase (EC 1.1.1.27) before a reaction with NADH. The degradation of pyruvate via NADH is measured by an extinction acceptance reaction at 340 nm, which can be directly correlated with the creatinine concentration in the sample.
Another procedure for creatinine detection in a sample includes the enzymatic conversion of creatinine by creatininase (EC 3.5.2.10.) to glycine, formaldehyde, and H2O2 (see FIG. 3).
This process requires two auxiliary reactions involving creatinase (EC 3.5.3.3.) and sarcosinoxidase (EC 1.5.3.1.). In a subsequent detection reaction, the increase in H2O2 formation is measured, upon the addition of peroxidase (EC 1.111.7.), via an extinction increase at 510 or 546 nm (Guder, W. G., Hoffman, G. E., Poppe, W. A., Siedel, J., Price, C. P. (1986). J. Chem. Clin. Biochem. 24: 889–902).
A further procedure for determining creatinine concentration is based on the creatinine deiminase (EC 3.5.4.21) catalyzed cleavage of creatinine to n-methylhydantoin and ammonia (Szulmajster, J. (1958). J. Bacteriol. 75: 633–639). The concentration of formed ammonia can be determined via a multilayer film technology using an indicator (Shirey, T. L. (1983). Clin. Biochem. 16: 147–152). Alternatively, the ammonia concentration can be determined as the ammonia reacts with α-ketoglutarate and NADPH/H+ in the presence of glutamate dehydrogenase to form glutamate, which can be measured photometrically via the extinction acceptance reaction at 340 and/or 365 nm (Lim, F. (1974). Clin. Chem. 20: 871; Tanganelli, E., Principe, L., Bassi, D., Cambiaghi, S., Murador, E. (1982). Clin. Chem. 28: 1461–1464; see FIG. 4).
The above-described enzymatic procedures for measuring creatinine concentration in a sample are generally not subject to the same interfering factors associated with the Jaffé method. However, these procedures fail to specifically measure only the creatinine concentration due to the presence of nonspecific substrates competing for creatinine deiminase in the catalytic reaction. For example, because both cytosine and all cytosine derivatives can effectively act as substrates for creatinine deiminase during the formation of creatinine, this necessarily results in an artificially high measurement of creatinine concentration in any particular sample where cytosine is present.